Viral Tissue Distribution,Histopathology And Its Apoptotic Effects In Ducklings Infected By Duck Circovirus

It is found that duck circovirus (DuCV) is the the smallest duck virus currently, the symptoms of ducklings infected by DuCV are growth retardation, weight loss and other symptoms. To date, the pathogenic mechanism of DuCV is not fully understood, circovirus infection commonly associated with lymphoid tissue damage, and apoptosis of lymphocytes can be caused. Therefore, the study for viral tissue distribution, histopathology and its apoptotic effects in ducklings infected by DuCV is of great scientific significance to discuss the pathogenic mechanism.1. The development of SYBR Green I-based quantitative PCR for detection of Duck circovirus and determination of the median infective doseIn order to the quantitative detection of infection distribution for DuCV in infected ducklings, the primers of DuCV-rep gene was designed according to the conserved region of DuCV-rep gene latest published in NCBI, and SYBR Green I-based quantitative PCR (qPCR) for detection of Duck circovirus was established. A series of experiments for evaluations of the reproducibility, sentitiviry, and specificity of the assay were performed. It can be achieved that there is no cross-reaction occurred with nucleic acids extracted from Riemerella anatipestifer (RA), Escherichia coli (E.coli) and other viruses as control templates; and maximal range of assay is 9.23×101copies/μL, the SD for Cq value of standard plasmid in different density in the range of 0.16-0.30, and CVs in the range of 0.79-1.64. In addition, median infective dose of positive DuCV material in suspension was detected (6.57 × 102.68 copies/mL) according to Reed-Muench method. Positive DuCV material in suspension was prepared, which was absolute quantitated by qPCR, and it was used for artificial infection of 3-week-old ducklings after the copy number of different virus dilution was carried out.2. SYBR Green I-based quantitative PCR for detection of viral tissue distribution and histopathology in ducklings infected by Duck circovirusTo detection of viral tissue distribution and histopathology in ducklings infected with Duck circovirus, histopathological observation of thymuses, spleens, bursas of Fabricius, livers, and kidneys of infected ducklings was carried out. Morever, SYBR Green I-based quantitative PCR for detection of viral tissue distribution in DuCV infected ducklings were performed. Histopathological examination showed that there were different degrees of pathological lesions in thymuses, spleens, bursas of Fabricius, livers, and kidneys, mainly including capillary congestion, hemorrhage and inflammatory cell infiltration; sparse lymphocytes, the gap of cells widening; macrophage infiltration; pyknosis and karyorrhexis of lymphocytes; vacuolar degeneration occurred and so on. qPCR results showed that DuCV can be detected in immunity organs, digestive organs, gastrointestinal tracts, respiratory tracts (except for the lungs) and other tissues at 8 dpi. Among of them, copies of viral genomes were 1035 copies-1045 copies in immunity organs,1039 copies-1046 copies in digestive organs,1028 copies-104.7 copies in gastrointestinal tracts,102.8 copies-104.1 copies in respiratory tracts, and 1026 copies-104-9 copies in other tissues.3. Dynamic of cell apoptosis in artificially infected ducklingsIn this study, immunohistochemistry technique and DNA terminal-deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) were applied in detection for antigen localization, antigen quantify and apoptosis in bursas of Fabricius, harderian glands, thymuses, spleens, livers, kidneys. Immunohistochemical results showed that average optical density was range from 0.0259±0.0148 to 0.1681 ±0.027 at 8dpi-48dpi and subsequently, a remarkable reducing (P<0.01) of DuCV antigen occurred in bursa of fabricius, harder gland, thymus, spleen, liver and kidney, and it suggested that DuCV replication was more active at 8dpi-48dpi. Apoptosis detection revealed that apoptosis response in experimental groups were more significant than control groups (P< 0.01). It was ambiguous that the relationship between virus load and apoptosis from those results, therefore it was incompletely to clarificate the relationship both of them. However, it suggested that DuCV can indeed cause target cell apoptosis significantly, in particular, it would cause immune organ damage in immunologic tissue and furthermore may induce immunosuppression. This paper focused on the relationship between DuCV infection between apoptosis, and it would be provide new ideas for us to detection, diagnosis, prevention and treatment and pathogenic mechanism of DuCV infection.